An Investigation into the effect of surface area on the activity of catalase in potato.

Preliminary work:

For my real experiment I have completed a pilot study, which has enabled me to test my choices of apparatus and methods that I will use, so that any alterations can be made in the event of there being a problem.

        During this preliminary study I have encountered a number of consequential problems. One of these involved the container in which to hold my experiment within, being that of a conical flask. However, through the pilot study I found that the size and volume of the conical flask affected the correct working of the glass syringe. It appeared that because of the volume of the conical flask, that which did not contain the hydrogen peroxide and potato chip(s), the pressure required for the glass syringe to work correctly was not present, and so would become jammed. Which after certain periods of time when the pressure of the oxygen gas being released got to a certain point would expel the glass syringe to jump up 2 or 3 milliliters at a time. Which as can be imagined, would cause the results collected to be very unreliable and extremely inaccurate. So on that point I had that I would use a smaller conical flask so solve the problem with a smaller volume, but on investigation found out that this piece of apparatus was not available to me. So in review of this information I compromised by instead using a boiling tube in the conical flasks place. Which was not a perfect or at all ideal choice, as when I would experiment upon more that one piece of potato chip in the investigation into the effect of surface area on the activity of catalase in potato, the surface area would be considerably affected at times due to the individual pieces of potato chip touching each other. Resulting in the calculated surface area, not being the actual surface area within the experiment. With reference to this, each method of either using the conical flask or boiling tube has its limitations, but I have come to the conclusion that although the surface areas may be jeopardized, at least I will have smooth and consistent results to read from is I use the boiling tube. However, because of the boiling tubes restrictions and limitations I will watch, and account for, whenever possible, the problem of the potato chips touching, being aware of these problems tied with using this method when writing up my evaluation.

        Other problems which I encountered, though not as dramatic or major, included reducing my volume of hydrogen peroxide used to 20ml from 100ml, because of the transaction from a conical flask to a boiling tube. However, after this adjustment I readjusted the measurement again to 15ml from 20ml, due to the hydrogen peroxide bubbling and foaming so much in the boiling tube that it started going up the delivery tube. As this would thereby affect my results in the collection of oxygen being released from the reaction taking place, I reduced the amount used. I also discovered that in order for the glass syringe to move freely with as little friction as possible, it was best to remove the inside of the syringe and wipe it all over with a clean cloth. This was in order to remove any moisture that may have clung to it, due to water being the other product being produced by the interaction between the hydrogen peroxide substrate molecule, and the active site of the catalase molecule.

Fair Test: 

Apparatus: 

For the experiment I will require:

• Goggles
• A Boiling tube
• A large Potato
• Borer – 8milimeters in diameter
• 15mililetres of hydrogen peroxide at 6% concentration and pH 6 per experiment undertaken
• 100 millilitres glass syringe
• Scalpel
• Tile
• A glass delivery tube
• A rubber bung to fit boiling tube
• Ruler (with measurements in millimetres)
• Stopwatch
• Test-tube/boiling-tube rack
• Thermostatic water bath with water set to 38°C or above
• 250 millilitres glass beaker
• Plastic gloves
• Rubber tube
• 25 millilitres measuring cylinder
• 3 millilitres plastic syringe
• Clamp
• Boss
• Stand

Equipment Justification table:

Method:

1. Set up apparatus as illustrated in diagram.
2. Measure out 15ml of hydrogen peroxide from a beaker into a 25ml measuring cylinder. At first pour in hydrogen peroxide up to approximately 13ml, then refer to use 3ml plastic syringe to get the meniscus of the hydrogen peroxide exactly on the line of 15ml.
3. Pour precisely measured out hydrogen peroxide from measuring cylinder, into boiling tube.
4. Place the boiling tube into a thermostatic water bath, using a thermometer within the boiling tube to measure the temperature of the hydrogen peroxide, until it is 38°C.
5. Using an 8mm borer, bore out a potato chip from a potato on the tile. Then using a scalpel remove the fibrous skin of the potato chip on the surface of the tile, making sure that it’s a straight, vertical incision.
6. Using a ruler, measure the potato chip out to 40mm and cut a vertical incision using the scalpel on the tile.
7. Fill a 250ml beaker with water at the temperature of 38°C using a thermometer. Proceed to place the boiling tube into the beaker. This will act an external water bath for regulating the hydrogen peroxide at 38°C.
8. Once temperature of hydrogen peroxide and its external water bath are at 38°C, place the potato chip into the solution of hydrogen peroxide, making sure that the syringe is set at 0ml.
9. As soon as potato chip is in solution of hydrogen peroxide, and bung has been securely inserted, start the stopwatch.
10. Readings of oxygen volume in syringe should be taken every 30seconds for 5minutes, and results recorded to results table.
11. Repeat steps 2-9 twice more, in order to lower the possibility of there being anomalous results.
12. Repeat steps 2-10 changing the potato chip surface area in step 5 to – two 20mm, four 10mm and eight 5mm pieces of potato chip. Resulting in four surface areas being investigated, and all results collected for correlation in graphs (Refer to diagram below to reference of how to cut potato chips).

Diagram

Safety:

• Care should be taken when handling the scalpel and cork borer, in order to avoid injury, as they are very sharp objects.

• Care should also be taken whilst handling the chemicals, by wearing plastic glove’s as hydrogen peroxide is an acidic corrosive, causing irritating burns on the surface of the skin that it comes into contact with. Hydrogen peroxide also has a side affect of staining any skin it comes into contact with, producing a white patch of dead tissue.
• Goggles should be worn to protect eyes from splashes of hydrogen peroxide, which if in contact with eyes rinse thoroughly with warm water.
• All stools should be tucked under tables to avoid accidents of tripping, whilst carrying out experiment, or walking around people who are. This is because there will be many hazardous pieces of equipment lying around the work benches, such as scalpels, borers and beakers full of hydrogen peroxide, which may cause serious injury to yourself, or someone you may fall into.
• All individuals with long hair should tie and clip it back in order to avoid it coming into contact with any hydrogen peroxide being used, as it may cause the hair to be bleached slightly.

Results Table:

Conclusion:

- See graphs attached
 

Interpretation:

The results show, in accordance with my prediction, a trend forming for when I increase the surface area of the potato chip, the reaction rate of catalase reacting with the hydrogen peroxide has increased. Thus, causing more oxygen to be produced, so that the collection of oxygen in the glass syringe to be much greater. This can be clearly observed in my graph showing 'the relationship between time and volume of oxygen collected' from the four surface areas that were experimented upon, by the increasing curves of the lines. This is because as the surface area of the potato chip increases, there is more of the enzyme catalase being exposed to the substrate molecule of hydrogen peroxide. Meaning that there are more enzyme molecules available to bind with the substrate molecules of hydrogen peroxide; causing the reaction to speed up. There is however, a large exception to this trend in the collection of my results where the surface areas named 2 and 3 fit the opposite way around to what I have predicted will happen. Where the smaller the surface area is, the greater the rate of reaction presents itself than that of the larger surface area. If it weren’t for these two surface areas being between the other two I maybe justified in saying that those were incorrect. Though being that they aren’t, with the other two in logical and predicted accordance with my prediction and scientific research and knowledge, it can be safe to say that surface areas 2 and 3 are the two sets of data which can be seen as anomalous results. However, it has to be kept in mind that the irregularity of the results may not be the cause of an error, but of something different, which I will investigate further in my evaluation of the experiment.

Although the idea of increased surface area being relational to the amount of oxygen released holds true, the two are not directly proportional. Where as I have previously mentioned these collations of results on the graph are not displayed as straight lines, but rather instead as curves. For each of the surface areas it is the same format, although at different values on the graph, where the curve starts off steep but gradually smoothes out as the experiment progresses through. This gradual deceleration of the rate of reaction is due to there being less substrate molecules to bind with the catalase enzyme molecules, and release the two products. This is clearly visible within my graphs, showing the volume of oxygen collected in the glass syringe, at 30-second intervals of the experiment, to slow, causing the curve to gently level off, reducing in its gradient.

With the last 30 seconds of the experiment I recorded, always being the point where the rate of reaction is at its least, and the initial rate of reaction being that of the greatest in the initial stages of the first 30 seconds. I believe that after these initial stages, the reaction will slow, due to there being less substrate molecules to bind with the catalase enzyme molecules, and release the two products. This is clearly visible within my graphs, where they show the volume of oxygen collected in the glass syringe, at 30-second intervals of the experiment, to slow, causing the curve to gently level off, reducing in its gradient.

This is because at the beginning of the experiment, the ‘Initial rate of reaction’ for catalase and hydrogen peroxide will be much faster. This is due to there being more substrate molecules available at this time to react with the enzyme molecules active site, causing the release of the two products; water and oxygen. The theoretical maximum rate of reaction, if all the factors affecting the reaction rate of catalase with hydrogen peroxide were at their optimum, would be at this particular stage of the reaction. This theoretical maximum is when all the sites are being used, but in reality this theoretical maximum is never reached due to the fact that not all the active sites are being used all the time. As all the substrate molecules need time to join onto the enzyme and to leave it so the maximum rate achieved is always slightly below the theoretical maximum. The time taken to fit into and leave the active site is the limiting factor in the rate of reaction. Though obviously the closer I can get to applying the optimum factors of catalase, the closer I will become in realising the theoretical maximum.

To investigate this first principle of initial rate of reaction, I drew a graph of all the surface areas' initial reaction rates from 0-30 seconds at the start of the reaction. Due to recording my results every 30 seconds I had no other option when doing this, other than to draw the results in a straight line from 0 to the volume of oxygen collected at the first reading of 30 seconds. From this I found that, as expected, the greater the surface area, the higher the rate of reaction. This also proved true for my two sets of ‘anomalous’ data, proving that in the first 30s seconds the reaction was working correctly in accordance with the results and trend that I have previously mentioned and specified upon.

I also went a little further with this idea, and compiled graphs of each of the four surface area showing their initial reaction rate, the first 30 seconds of the experiment, and their final reaction rate, the last 30 seconds of the experiment. This was so that I could compare and analyse the two graphs in front of me, as well as working out the gradients of two straight lines, so that the change in gradient from the beginning to the end could be found.

What I found was that the smallest of the surface areas (surface area 1), undergone the least amount of change in its gradient, being that of 0.04. With surface area 2 at 0.05, surface area 3 at 0.066, and surface area 4 with the largest surface area and greatest change in gradient being 0.07. Giving pattern to the fact that the larger the surface area, the greater the change in the gradient will be overall, through the entire duration of the experiment.  

To conclude, therefore, that the results I have obtained from carrying out the experiment, support my initial prediction, enabling me to discover that varying the surface areas of the potato chip, is a large and considerable factor in the effect of surface area on the activity of catalase in potato.

Evaluation:

To help make this experiment more accurate, I repeated it three times and then used the average of all the results to plot a graph with a line of best fit. I tried to keep all the variables the same, except for the surface area of the potato chip, the same for all the experiments. However, in reality it is impossible to keep all the variables precisely the same. For example:

• There is a slight delay between placing the potato chip in the hydrogen peroxide solution, putting the bung on and starting the stopwatch. This will slightly affect all the results, as oxygen produced from reaction would be lost in this space of time, but as I carried out all the three steps in the same way for all the experiments it should not make any difference to the overall result.
• The slight delay between transporting the heated water bath and boiling tube containing hydrogen peroxide, both set at 38°C exactly, to inserting the potato chip, inserting the bung and then starting the stopwatch, allowed time for the temperature of 38°C to drop. This will slightly affect all the results, as the enzyme will start working in an environment just below that of 38 °C. But seeing as I carried out all the four steps in the same way for all the experiments it should not have made any difference to the overall result.

As a note if I were to do the experiment again: The temperature of the external water bath dropped by 4 °C and 5 °C for the hydrogen peroxide inside the boiling tube within the external water bath, by the end of the experiment.

• The volume of air in the test tube and tubing to start with will inevitably effect the collection of oxygen from the reaction. This is because when the bung is pushed down into the boiling tube, the gas within the boiling tube is compressed, forcing the air through into the tubing, as an attempt to relieve the pressure within the boiling tube. However, air being forced into the tubing, will result in the glass syringe filling with this air, in order to balance out the pressure, and so before the experiment truly begins there is a measurement of gas in the glass syringe.
• The factor of the chips touching may constitute largely in inaccuracy of the collection of whole sets of data, due to the calculated surface areas in theory, being incorrect and unreliable in practical. This is a very compromising situation as previously mentioned in the section headed ‘preliminary study’, where it was a choice between a boiling tube, or a conical flask to contain the experiment within. Both presenting limitations, with lack of pressure to move the glass syringe in a smooth movement being one for the conical flask, and the problem just described of the potato chips touching each other being that for the boiling tube idea. So in affect there was little I could do to limit this limiting factor in my choice of using a boiling tube. With the deciding point being that at least with a boiling tube my results would be smooth and flowing, and accurate to the times at which the measurements were taken.

• Through selection of the sizes of potatoes for use within the experiment, which was out of my control, I have had to resort to using two separate potatoes, due to the ones selected being too small to use for all my experiments. Though both potatoes were from the same bag, they may have vast differences between them when it comes to the distribution and concentration of catalase in the cells. This I believe was the main reason for the problem I occurred as scripted above in my interpretation, where two surface areas, known as surface area 2 and surface area 3, did not conform to the prediction, but instead did the complete opposite to it. Which in this case was where the smaller of the two surface areas having the greater of the rates of reaction, and vice versa in the larger potato chip. So that it appears, in affect, that surface area 2 on the graph of ‘the relationship between time and volume of oxygen collected', should be surface area 3 and the other way around. Which without explanation may be viewed as a typing error.

However, through analysing and interpreting my results, I have found that at the stage of the ‘initial rate of reaction’, the two surface areas conform to my prediction, where in simple terms the greater the surface area of the potato chip, the greater the production of oxygen will be from the reaction. I also found to be true, that the change in gradients for the two surface areas, within the results, throughout the experiment from initial rate of reaction to final rate of reaction, was that the larger of the two surface areas (surface area 3), was found to reduce faster in the rate of reaction through the measurement of gradient, than that of surface area 2. Meaning that the anomaly of the two sets of results is due to a change in the level of readings being taken of the collection of oxygen. Which I believe is due to the fact that for surface areas 1 and 2, one potato was used, but then for surface areas 3 and 4 another different potato was used for all the results. This was due to the fact that no more complete bores of potato chips could be made from the original potato. Constituting in a possible change of catalase concentration within the 2nd potato. Though this would only be a moderate change in the catalase concentration within the potato chips, and so would not affect the initial reaction rate as proven in my graphs. My theory upon this is that the potato used for surface area 3 and 4, with the larger surface area, was that of a lower catalase concentration than the potato used for S.A 1 and 2. Thereby, resulting in the results past the initial phase to be lower than that of S.A 2. But however, in the initial reaction rate stage the results are proportional to the prediction, as it should have been if the same potato was used throughout all 4 surface areas. As although the potato chip catalase concentration was lower, its surface area was greater, causing the initial reaction rate to balance out in the correct manner, with S.A’s 3 results possessing a faster rate of reaction than that of S.A 2. It can be seen however, that S.A 2 overtakes S.A 3 in its rate of reaction past this initial point. Which I believe is because the amount of catalase being released from the cells of the potato chip in S.A 3 carries on in its lower release speed, to that in comparison to the potato being used for S.A 2. Thus, allowing the potato chip of S.A 2 to overtake in the rate of reaction of S.A 3.

For future studies and reference, it would be useful to take this observation and theory further by graphing the relationship between surface area and the release of catalase from the potato chips for the two surface areas and potatoes. Analysing where the two lines meet, and how this affects the results of this experiment.

This experiment could be improved in a number of ways. It could be repeated more times to help get rid of any anomalies. A better overall result would be obtained by repeating the experiment more times because any errors in one experiment should be compensated for by the other experiments.

Using more surface areas of potato chips would have produced more data to draw from when examining it against my prediction, or any other acclaimed theories upon the reaction rate of catalase in potato and hydrogen peroxide. .

The problem of the delay between dropping in the potato chip, bunging the test tube and starting the stopwatch could have been limited by getting another person to start the stopwatch when the potato chip was dropped into the boiling tube.

By recording the reaction for longer than 5 minutes in order to gather more data to make a more informed judge of what is happening. Also, to take the measurements every 10 seconds, instead of every 30 seconds so that the results are more accurate.

Overall, I believe the experiment was fairly reliable, as I obtained smooth and reliable results. Finding that as long as all the variables that were designed to be kept constant, i.e. the potato used throughout, actually were, then the main problems I accoutered would not have caused a problem within the collection of my results in the ‘Investigation into the effect of surface area on the activity of catalase in potato’.

Bibliography

Websites:

1.  / December 2001
2.  / December 2001

5.  / December 2001

Textbooks:

3. Jones, M., Fosbery, R. and Taylor, D. (2000) Advanced Sciences Biology 1, Cambridge. Page 42

CD-ROMs:

4. "Structure and Function of an Enzyme," Microsoft® Encarta® Encyclopedia 2000. © 1993-1999 Microsoft Corporation. All rights reserved.